研究胚胎损伤雏鸡角膜的无疤痕组织再生
Summary
本方案演示了在 卵中伤害胚胎雏鸡角膜所涉及的不同步骤。再生或完全恢复的角膜可以在伤口手术后使用各种细胞和分子技术分析再生潜力。
Abstract
雏鸡胚胎角膜伤口表现出完全和快速再生的显着能力，而成年受伤的角膜由于纤维化瘢痕形成而失去透明度。受伤胚胎角膜的组织完整性内在恢复，没有可检测到的疤痕形成。鉴于其可及性和易于操作性，雏鸡胚胎是研究无疤痕角膜伤口修复的理想模型。该协议展示了在 卵中伤害胚胎雏鸡角膜所涉及的不同步骤。首先，卵子在胚胎早期年龄开窗以进入眼睛。其次，对胚胎外膜进行一系列 卵内 物理操作，以确保在发育的后期阶段保持对眼睛的访问，对应于角膜的三个细胞层形成的时间。第三，穿透外上皮层和前基质的线性角膜伤口是使用显微外科刀制成的。在伤口手术之后，可以使用各种细胞和分子技术分析再生过程或完全恢复的角膜的再生潜力。迄今为止使用该模型的研究表明，受伤的胚胎角膜显示角质形成细胞分化的激活，ECM蛋白协调重塑为其天然的三维宏观结构，并被角膜感觉神经充分重新支配。将来，内源性或外源性因素对再生过程的潜在影响可以通过使用发育生物学技术（例如组织移植，电穿孔，逆转录病毒感染或珠子植入）来分析角膜愈合。目前的策略将胚胎雏鸡确定为阐明协调无疤痕角膜伤口愈合的分子和细胞因子的关键实验范例。
Introduction
角膜是眼睛最外层的透明组织，透射和折射有利于视力的光。在成人角膜中，角膜基质的损伤或感染导致快速而强大的伤口愈合反应，其特征在于角质形成细胞增殖，纤维化，导致细胞因子诱导的细胞凋亡的炎症增加，修复肌成纤维细胞的产生以及细胞外基质（ECM）的整体重塑1，2.损伤后，这种角膜组织修复导致不透明的疤痕组织，降低角膜透明度并阻塞光线通过，从而扭曲视力，在最严重的情况下，导致角膜失明3。因此，显然需要开发可靠的动物模型来解决伤口愈合的复杂性，并确定负责伤口闭合和组织再生的细胞和分子因素。
迄今为止，大多数检查角膜伤口愈合的研究都使用了产后4 或成年动物模型1，2，5，6，7。虽然这些研究在理解角膜伤口愈合反应和疤痕形成机制方面取得了重大进展，但这些愈合模型中受损的角膜组织未能完全再生，从而限制了它们识别负责完全概括损伤后角膜形态和结构的分子因素和细胞机制的实用性。相比之下，用刀在胚胎雏鸡角膜中产生的胎儿伤口具有以无疤痕的方式完全愈合的内在能力8。具体而言，胚胎雏鸡角膜表现出非纤维再生，细胞外基质结构和神经支配模式8，9的完全概括。
本方案描述了在 卵中伤害胚胎雏鸡角膜的一系列步骤。首先，卵子在胚胎早期年龄开窗，以促进胚胎的进入。其次，对胚胎外膜进行一系列 卵内 物理操作，以确保在发育的后期阶段保持对眼睛的访问，对应于角膜的三个细胞层形成并且需要伤口的时间。第三，使用显微外科刀制作穿透角膜上皮并进入前基质的线性中央角膜切口。在伤口手术之后，可以使用各种细胞和分子技术分析再生过程或完全恢复的角膜的再生潜力。
Protocol
该协议中使用的鸡蛋菌株是白色里窝那，所有动物程序都得到了伊利诺伊卫斯理大学机构动物护理和使用委员会的批准。 1. 雏鸡蛋的孵化 产卵后将卵保持在~10°C长达1周以停止发育。当准备开始雏鸡胚胎发育时，用室温水饱和的无绒湿巾（参见 材料表）擦拭整个蛋壳，以除去污垢和碎屑。 确保蛋壳经过消毒。用蘸有70%乙醇的无绒湿巾?…
Representative Results
在E5.5处对ACM和CAM进行早期解剖以暴露发育中的胚胎的颅骨区域之后， 在卵中 进行了一系列跨越E7中心角膜的撕裂伤（图1）。研究角膜再生的理想伤口发生在三次撕裂伤之后，每次撕裂伤都在角膜的同一位置进行。第一次撕裂伤穿过角膜上皮，而第二次和第三次撕裂分别穿透下面的基底膜和前基质。为了获得理想的伤口，使用锋利的微解剖刀（参见 材料表</stron…
Discussion
雏鸡是研究胎儿无疤痕角膜伤口修复的理想模型系统。与哺乳动物不同，在整个发育过程中，使用 卵8或 前卵 策略24很容易获得雏鸡。胚胎雏鸡角膜比啮齿动物角膜大得多，近50%的颅骨体积专用于眼睛25，使其非常适合物理操作，例如受伤。此外，鸡蛋全年都很容易买到，通常来自当地农场，而且具有成本效益，只需要一个温水?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了伊利诺伊卫斯理大学向TS提供的艺术和学术发展补助金的支持，部分资金来自NIH-R01EY022158（PL）。
Materials
| 18 G hypodermic needle | Fisher Scientific | 14-826-5D | |
| 30 degree angled microdissecting knife | Fine Science Tools | 10056-12 | |
| 4′,6-diamidino-2-phenylindole (DAPI) | Molecular Probes | D1306 | |
| 5 mL syringe | Fisher Scientific | 14-829-45 | |
| Alexa Fluor labelled secondary antibodies | Molecular Probes | ||
| Calcium chloride dihydrate (CaCl2-H20) | Sigma | C8106 | |
| Chicken egg trays | GQF | O246 | |
| Dissecting Forceps, Fine Tip, Serrated | VWR | 82027-408 | |
| Dissecting scissors, sharp tip | VWR | 82027-578 | |
| Iris 1 x 2 Teeth Tissue Forceps, Full Curved | VWR | 100494-908 | |
| Kimwipes | Sigma | Z188956 | |
| Microdissecting Scissors | VWR | 470315-228 | |
| Mouse anti-fibronectin (IgG1) | Developmental Studies Hybridoma Bank | B3/D6 | |
| Mouse anti-laminin (IgG1) | Developmental Studies Hybridoma Bank | 3H11 | |
| Mouse antineuron-specific β-tubulin (Tuj1, IgG2a) | Biolegend | 801213 | |
| Mouse anti-tenascin (IgG1) | Developmental Studies Hybridoma Bank | M1-B4 | |
| Paraformaldehyde | Sigma | 158127 | |
| Penicillin/Streptomycin | Sigma | P4333 | |
| Potassium chloride (KCl) | Sigma | P5405 | |
| Sodium chloride (NaCl) | Fisher Scientific | BP358 | |
| Sportsman 1502 egg incubator | GQF | 1502 | |
| Tear by hand packaging (1.88 inch width) | Scotch | n/a |
Referências
- Wilson, S. E. Corneal wound healing. Experimental Eye Research. 197, 108089 (2020).
- Ljubimov, A. V., Saghizadeh, M. Progress in corneal wound healing. Progress in Retinal and Eye Research. 49, 17-45 (2015).
- Whitcher, J. P., Srinivasan, M., Upadhyay, M. P. Corneal blindness: a global perspective. Bulletin of the World Health Organization. 79 (3), 214-221 (2001).
- Ritchey, E. R., Code, K., Zelinka, C. P., Scott, M. A., Fischer, A. J. The chicken cornea as a model of wound healing and neuronal re-innervation. Molecular Vision. 17, 2440-2454 (2001).
- Berdahl, J. P., Johnson, C. S., Proia, A. D., Grinstaff, M. W., Kim, T. Comparison of sutures and dendritic polymer adhesives for corneal laceration repair in an in vivo chicken model. Archives of Ophthalmology. 127 (4), 442-447 (2009).
- Fowler, W. C., Chang, D. H., Roberts, B. C., Zarovnaya, E. L., Proia, A. D. A new paradigm for corneal wound healing research: the white leghorn chicken (Gallus gallus domesticus). Current Eye Research. 28 (4), 241-250 (2004).
- Huh, M. I., Kim, Y. E., Park, J. H. The distribution of TGF-β isoforms and signaling intermediates in corneal fibrotic wound repair. Journal of Cellular Biochemistry. 108 (2), 476-488 (2009).
- Spurlin, J. W., Lwigale, P. Y. Wounded embryonic corneas exhibit nonfibrotic regeneration and complete innervation. Investigative Ophthalmology & Visual Science. 54 (9), 6334-6344 (2013).
- Koudouna, E., Spurlin, J., Babushkina, A., Quantock, A. J., Jester, J. V., Lwigale, P. Y. Recapitulation of normal collagen architecture in embryonic wounded corneas. Scientific Reports. 10 (1), 13815 (2020).
- Luo, J., Redies, C. Ex ovo electroporation for gene transfer into older chicken embryos. Developmental Dynamics. 233 (4), 1470-1477 (2005).
- Spurlin, J., Lwigale, P. Y. A technique to increase accessibility to late-stage chick embryos for in ovo manipulations. Developmental Dynamics. 242 (2), 148-154 (2013).
- Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chick embryo. Journal of Morphology. 88 (1), 49-92 (1951).
- Neath, P., Roche, S. M., Bee, J. A. Intraocular pressure dependent and independent growth phases of the embryonic chick eye and cornea. Investigative Ophthalmology & Visual Science. 32 (9), 2483-2491 (1991).
- Matsuda, A., Yoshiki, A., Tagawa, Y., Matsuda, H., Kusakabe, M. Corneal wound healing in tenascin knockout mouse. Investigative Ophthalmology & Visual Science. 40 (6), 1071-1080 (1990).
- Nishida, T., Nakagawa, S., Nishibayashi, C., Tanaka, H., Manabe, R. Fibronectin enhancement of corneal epithelial wound healing of rabbits in vivo. Archives of Ophthalmology. 102 (3), 455-456 (1984).
- Sumioka, T., et al. Impaired cornea wound healing in a tenascin C-deficient mouse model. Lab Investigation. 93 (2), 207-217 (2013).
- Tervo, K., van Setten, G. B., Beuerman, R. W., Virtanen, I., Tarkkanen, A., Tervo, T. Expression of tenascin and cellular fibronectin in the rabbit cornea after anterior keratectomy. Immunohistochemical study of wound healing dynamics. Investigative Ophthalmology & Visual Science. 32 (11), 2912-2918 (1991).
- Lwigale, P. Y., Bronner-Fraser, M. Lens-derived Semaphorin3A regulates sensory innervation of the cornea. Biologia do Desenvolvimento. 306 (2), 750-759 (2007).
- Kubilus, J. K., Linsenmayer, T. F. Developmental corneal innervation: interactions between nerves and specialized apical corneal epithelial cells. Investigative Ophthalmology & Visual Science. 51 (2), 782-789 (2010).
- Schwend, T., Deaton, R. J., Zhang, Y., Caterson, B., Conrad, G. W. Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation. Investigative Ophthalmology & Visual Science. 53 (13), 8118-8137 (2012).
- Lee, M. K., Tuttle, J. B., Rebhun, L. I., Cleveland, D. W., Frankfurter, A. The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis. Cell Motility and the Cytoskeleton. 17 (2), 118-132 (1990).
- Chen, X., Nadiarynkh, O., Plotnikov, S., Campagnola, P. J. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nature Protocols. 7, 654-669 (2012).
- Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. J., Mohler, W. A. Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophysical Journal. 82 (1), 493-508 (2002).
- Cloney, K., Franz-Odendaal, T. A. Optimized ex-ovo culturing of chick embryos to advanced stages of development. Journal of Visualized Experiments. (95), e52129 (2015).
- Waldvogel, J. A. The bird’s eye view. American Scientist. 78, 342-353 (1990).
- Martin, P., Parkhurst, S. M. Parallels between tissue repair and embryo morphogenesis. Development. 131 (13), 3021-3034 (2004).
- Wilson, S. E., Mohan, R. R., Mohan, R. R., Ambrosio, R., Hong, J., Lee, J. The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Progress in Retinal and Eye Research. 20 (5), 625-637 (2001).
Citar este artigo
Pathuri, M., Spurlin III, J., Lwigale, P., Schwend, T. Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas. J. Vis. Exp. (183), e63570, doi:10.3791/63570 (2022).